The stage-specific testicular germ cell apoptotic response to low-dose X-irradiation and 2,5-hexanedione combined exposure. I: Validation of the laser capture microdissection method for qRT-PCR array application

Abstract

Over the past decade, laser capture microdissection (LCM) has grown as a tool for gene expression profiling of small numbers of cells from tumor samples and of specific cell populations in complex tissues. LCM can be used to study toxicant effects on selected cell populations within the testis at different stages of spermatogenesis. There are several LCM-related hurdles to overcome, including issues inherent to the method itself, as well as biases that result from amplifying the LCM-isolated RNA. Many technical issues associated with the LCM method are addressed here, including increasing RNA yield and obtaining more accurate quantification of RNA yields. We optimized the LCM method optimized to generate RNA quantities sufficient for quantitative reverse transcription polymerase chain reaction (qRT-PCR) array analysis without amplification and were able to validate the method through direct comparison of results from unamplified and amplified RNA from individual samples. The addition of an amplification step for gene expression studies using LCM RNA resulted in a bias, especially for low abundance transcripts. Although the amplification bias was consistent across samples, researchers should use caution when comparing results generated from amplified and unamplified LCM RNA. Here, we have validated the use of LCM-derived RNA with the qRT-PCR array, improving our ability to investigate cell-type and stage-specific responses to toxicant exposures.

Keywords: PCR array.; amplification; laser capture microdissection; testis.

Figure 1

Laser capture microdissection method schematic. For 1 sample replicate, about 20 seminiferous tubules from 12 slides were microdissected. The polymer film from each cap was peeled off and placed into 600 μl of mirVana Lysis/Binding buffer until all slides had been microdissected. After the final film had been added, the film/buffer mixture was incubated on ice for 45 min, following which RNA was extracted using the mirVana kit. This process was repeated 7 times and then each replicate is DNase treated, then combined and concentrated using the RNeasy MinElute Kit yielding approximately 350 ng of RNA.

Figure 2

Micrograph of an intact rat seminiferous tubule before LCM (A), the remaining tissue following LCM (B), and the isolated tissue on the LCM cap (C). Scale bar is 50 μm.

Figure 3

LCM-derived seminiferous tubule RNA quality assessment. Digital gel (A) and electropherogram results (B–C) obtained with the Agilent 2100 Bioanalyzer. Electropherogram results are shown for before (B) and after (C) DNase treatment and RNA concentration, with RINs of 5.40 and 6.50, respectively.

Figure 4

Effect of amplification on measured gene expression. Ratio of mean (and standard deviation) of amplified to unamplified mean cycle ratios across genes using sample data from both our lab and SABiosciences. Genes highlighted in black, Traf3, Tp53, Faslg, Dffb, Bcl2a1d and Bcl2l11, have a high standard deviation.

Figure 5

Consistency of RNA input method across samples. Unamplified and amplified ΔCt value comparison. Graphs are shown comparing the ΔCt values for amplified (A) and unamplified (B) control biological replicates from our lab and amplified (C) and unamplified (D) 0.33% HD exposed sample data generated by SABiosciences.

Figure 6

Consistency of RNA input method within samples. The unamplified and amplified ΔCt values were compared within samples for a control sample from our lab (A) and a 0.33% HD exposed sample from SABiosciences (B). The data points highlighted in red (A) indicate outliers in the correlation and the corresponding genes for the data points are labeled.

Figure 7

Effect of RNA input concentration on threshold cycle values. (A) The threshold cycles for 14 ng of amplification input RNA compared to input RNA values of 7 ng (blue line), 35 ng (green line) and 98 ng (purple line). (B) The cycle threshold values for each gene at each amplification input RNA concentration, 7 ng (blue line), 14 ng (pink line), 35 ng (green line) and 98 ng (purple line). Highlighted genes, Tnfsf10, Bnip1, Bnip2, Dffb, Bcl2a1d, and Bcl2l11, indicate where the Ct values are not consistent across the different RNA values.